title: Characterization of a newly identified mycobacterial tautomerase with promiscuous dehalogenase and hydratase activities reveals a functional link to a recently diverged cis-3-chloroacrylic acid dehalogenase.
abstract: The enzyme cis-3-chloroacrylic acid dehalogenase (cis-CaaD) is found in a bacterial pathway that degrades a synthetic nematocide, cis-1,3-dichloropropene, introduced in the 20th century. The previously determined crystal structure of cis-CaaD and its promiscuous phenylpyruvate tautomerase (PPT) activity link this dehalogenase to the tautomerase superfamily, a group of homologous proteins that are characterized by a catalytic amino-terminal proline and a β-α-β structural fold. The low-level PPT activity of cis-CaaD, which may be a vestige of the function of its progenitor, prompted us to search the databases for a homologue of cis-CaaD that was annotated as a putative tautomerase and test both its PPT and cis-CaaD activity. We identified a mycobacterial cis-CaaD homologue (designated MsCCH2) that shares key sequence and active site features with cis-CaaD. Kinetic and 1H NMR spectroscopic studies show that MsCCH2 functions as an efficient PPT and exhibits low-level promiscuous dehalogenase activity, processing both cis- and trans-3-chloroacrylic acid. To further probe the active site of MsCCH2, the enzyme was incubated with 2-oxo-3-pentynoate (2-OP). At pH 8.5, MsCCH2 is inactivated by 2-OP due to the covalent modification of Pro-1, suggesting that Pro-1 functions as a nucleophile at pH 8.5 and attacks 2-OP in a Michael-type reaction. At pH 6.5, however, MsCCH2 exhibits hydratase activity and converts 2-OP to acetopyruvate, which implies that Pro-1 is cationic at pH 6.5 and not functioning as a nucleophile. At pH 7.5, the hydratase and inactivation reactions occur simultaneously. From these results, it can be inferred that Pro-1 of MsCCH2 has a pKa value that lies in between that of a typical tautomerase (pKa of Pro-1∼6) and that of cis-CaaD (pKa of Pro-1∼9). The shared activities and structural features, coupled with the intermediate pKa of Pro-1, suggest that MsCCH2 could be characteristic of an evolutionary intermediate along the past route for the divergence of cis-CaaD from an unknown superfamily tautomerase. This makes MsCCH2 an ideal candidate for laboratory evolution of its promiscuous dehalogenase activity, which could identify additional features necessary for a fully active cis-CaaD. Such results will provide insight into pathways that could lead to the rapid divergent evolution of an efficient cis-CaaD enzyme.
authors.affiliation: Department of Pharmaceutical Biology, Groningen Research Institute of Pharmacy, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands.
authors.forename: Bert-Jan
authors.initials: BJ
authors.lastname: Baas
authors.forename: Ellen
authors.initials: E
authors.lastname: Zandvoort
authors.forename: Anna A
authors.initials: AA
authors.lastname: Wasiel
authors.forename: Wim J
authors.initials: WJ
authors.lastname: Quax
authors.forename: Gerrit J
authors.initials: GJ
authors.lastname: Poelarends
mesh_terms: D000179:Acrylates / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D055162:Biocatalysis; D020134:Catalytic Domain; D005231:Fatty Acids, Unsaturated / Q000737:chemistry / Q000378:metabolism; D006836:Hydro-Lyases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006867:Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019746:Intramolecular Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008956:Models, Chemical; D008969:Molecular Sequence Data; D015394:Molecular Structure; D020102:Mycobacterium smegmatis / Q000201:enzymology* / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013237:Stereoisomerism; D013379:Substrate Specificity
chemical_list: C109822:2-oxo-3-pentynoic acid; D000179:Acrylates; D001426:Bacterial Proteins; D005231:Fatty Acids, Unsaturated; C071773:3-chloroacrylic acid; D006867:Hydrolases; C077719:cis-3-chloroacrylic acid dehalogenase; D006836:Hydro-Lyases; D019746:Intramolecular Oxidoreductases; C022346:phenylpyruvate tautomerase